Cancer is a disease of the genome. Identification of the genetic and epigenetic changes, such as aberrant DNA mutations and modifications underlying the development of carcinogenesis should provide unambiguous detection of cancer. These molecular changes, in concert with the genetic makeup of a patient, determine the clinical phenotype of the tumor, its response to a given treatment, and the patient's prognosis.
Hepatocellular carcinoma (HCC) is the most frequent cancer in certain parts of the world, and the fifth most cancer common worldwide. Increased incidence of HCC is associated with infections of hepatitis B virus and hepatitis C virus (El-Serag & Mason, 1999). Surgical resection is often curative if the disease is localized and diagnosis occurs early (Klintmalm, 1998). However, diagnosis of HCC is often late, resulting in extensive liver impairment associates with a poor prognosis, often due to late diagnosis. Late diagnosis of HCC is due to limited indicators. For example, among patients with underlying cirrhotic disease, a progressive increase in alpha-fetoprotein (AFP) and/or in alkaline phosphatase or a rapid deterioration of hepatic function may be the only clue to the presence of cancer.
Despite the availability of a preventative vaccine, chronic HBV infection remains a global health issue affecting more than 3500 million cases of HBV-related HCC (HBV-HCC). The HBV genome has 2-3 typical CpG islands depending on the genotype (Vivekanandan, Thomas, & Torbenson, 2008; Y. Zhang et al., 2013). Interestingly, these CpG islands are located at strategic locations in the regulatory elements of the HBV genome. For example, CpG island 1 is located in the first exon start site for the S (surface antigen) gene, and CpG islands 2 and 3 cover the enhancer II and the promoter of pregenomic RNA and the first exon start site of the polymerase gene, respectively (Moolla, Kew, & Arbuthnot, 2002; Vivekanandan et al., 2008). Although the virion DNA was found to be mostly unmethylated in both tissue culture and patient serum (Kaur et al., 2010; Vivekanandan et al., 2008), DNA methylation of the intranuclear HBV genome has been associated with repression of gene transcription in cultures (Guo, Li, Mu, Zhang, & Yan, 2009; Kim et al., 2011; Zhang, Hou, & Lu, 2013). Despite the difficulty of dissecting HBV DNA in diseased tissues, higher levels of methylation of CpG islands were found in HCC tissues compared to hepatitis and cirrhosis tissues, thus, methylation of HBV DNA has been associated with hepatocarcinogenesis.
Current methods of detection of HBV DNA genome and methylated HBV DNA are either not available or insensitive for fragmented DNA such as formalin fixed paraffin embedded (FFPE) tissues, DNA isolated from body fluids, merely qualitative. There remains a need for assays to quantitatively measure for the HBV DNA and its modifications (methylation) that can be used to monitor for the presence of HBV DNA and its modifications that are related to HCC and to screen for HCC so that HCC can be detected early and administered early.